Specific T-cell subsets have a role in anti-viral immunity and pathogenesis but not viral dynamics or onwards vector transmission of an important livestock arbovirus.

Introduction: Bluetongue virus (BTV) is an arthropod-borne Orbivirus that is almost solely transmitted by Culicoides biting midges and causes a globally important haemorrhagic disease, bluetongue (BT), in susceptible ruminants. Infection with BTV is characterised by immunosuppression and substantial lymphopenia at peak viraemia in the host. Methods: In this study, the role of cell-mediated immunity and specific T-cell subsets in BTV pathogenesis, clinical outcome, viral dynamics, immune protection, and onwards transmission to a susceptible Culicoides vector is defined in unprecedented detail for the first time, using an in vivo arboviral infection model system that closely mirrors natural infection and transmission of BTV. Individual circulating CD4+, CD8+, or WC1+ γδ T-cell subsets in sheep were depleted through the administration of specific monoclonal antibodies. Results: The absence of cytotoxic CD8+ T cells was consistently associated with less severe clinical signs of BT, whilst the absence of CD4+ and WC1+ γδ T cells both resulted in an increased clinical severity. The absence of CD4+ T cells also impaired both a timely protective neutralising antibody response and the production of IgG antibodies targeting BTV non-structural protein, NS2, highlighting that the CD4+ T-cell subset is important for a timely protective immune response. T cells did not influence viral replication characteristics, including onset/dynamics of viraemia, shedding, or onwards transmission of BTV to Culicoides. We also highlight differences in T-cell dependency for the generation of immunoglobulin subclasses targeting BTV NS2 and the structural protein, VP7. Discussion: This study identifies a diverse repertoire of T-cell functions during BTV infection in sheep, particularly in inducing specific anti-viral immune responses and disease manifestation, and will support more effective vaccination strategies.

Visualisation of Bluetongue Virus in the Salivary Apparatus of Culicoides Biting Midges Highlights the Accessory Glands as a Primary Arboviral Infection Site.

BACKGROUND: Arthropods transmit a wide range of pathogens of importance for the global health of humans, animals, and plants. One group of these arthropod vectors, Culicoides biting midges (Diptera: Ceratopogonidae), is the biological vector of several human and animal pathogens, including economically important livestock viruses like bluetongue virus (BTV). Like other arthropod-borne viruses (arboviruses), Culicoides-borne viruses must reach and replicate in the salivary apparatus, from where they can be transmitted to susceptible hosts through the saliva during subsequent blood feeding. Despite the importance of the salivary gland apparatus for pathogen transmission to susceptible animals from the bite of infected Culicoides, these structures have received relatively little attention, perhaps due to the small size and fragility of these vectors. RESULTS: In this study, we developed techniques to visualize the infection of the salivary glands and other soft tissues with BTV, in some of the smallest known arbovirus vectors, Culicoides biting midges, using three-dimensional immunofluorescence confocal microscopy. We showed BTV infection of specific structures of the salivary gland apparatus of female Culicoides vectors following oral virus uptake, related visualisation of viral infection in the salivary apparatus to high viral RNA copies in the body, and demonstrated for the first time, that the accessory glands are a primary site for BTV replication within the salivary apparatus. CONCLUSIONS: Our work has revealed a novel site of virus-vector interactions, and a novel role of the accessory glands of Culicoides in arbovirus amplification and transmission. Our approach would also be applicable to a wide range of arbovirus vector groups including sand flies (Diptera: Psychodidae), as well as provide a powerful tool to investigate arbovirus infection and dissemination, particularly where there are practical challenges in the visualization of small size and delicate tissues of arthropods.

Emergence dynamics of adult Culicoides biting midges at two farms in south-east England.

BACKGROUND: Culicoides biting midges (Diptera: Ceratopogonidae) are biological vectors of livestock arboviruses that cause diseases with significant economic, social and welfare impacts. Within temperate regions, livestock movement during arbovirus outbreaks can be facilitated by declaring a 'seasonal vector-free period' (SVFP) during winter when adult Culicoides are not active. In this study we carry out long-term monitoring of Culicoides adult emergence from larval development habitats at two farms in the UK to validate current definitions of the SVFP and to provide novel bionomic data for known vector species. METHODS: Standard emergence traps were used to collect emerging adult Culicoides from dung heaps at two cattle farms in the south-east of England from June to November 2016 and March 2017 to May 2018. Culicoides were morphologically identified to species or complex level and count data were analysed using a simple population dynamic model for pre-adult Culicoides that included meteorological components. RESULTS: More than 96,000 Culicoides were identified from 267 emergence trapping events across 2 years, revealing clear evidence of bivoltinism from peaks of male populations of Culicoides obsoletus emerging from dung heaps. This pattern was also reflected in the emergence of adult female Obsoletus complex populations, which dominated the collections (64.4% of total catch) and emerged throughout the adult active period. Adult male C. obsoletus were observed emerging earlier than females (protandry) and emergence of both sexes occurred throughout the year. Culicoides chiopterus and Culicoides scoticus were also identified in spring emergence collections, providing the first evidence for the overwintering of larvae in dung heaps for these species. CONCLUSIONS: This study demonstrates continual and highly variable rates of emergence of Culicoides throughout the year. A lack of evidence for mass emergence in spring along with the ability to observe male generations highlights the need for complementary surveillance techniques in addition to light-trap data when investigating seasonality and phenology. Evidence was found of other vector species, C. chiopterus and C. scoticus, utilising cattle dung heaps as an overwintering habitat, further highlighting the importance of these habitats on farms.

Field-Reassortment of Bluetongue Virus Illustrates Plasticity of Virus Associated Phenotypic Traits in the Arthropod Vector and Mammalian Host In Vivo.

Segmented RNA viruses are a taxonomically diverse group that can infect plant, wildlife, livestock and human hosts. A shared feature of these viruses is the ability to exchange genome segments during coinfection of a host by a process termed "reassortment." Reassortment enables rapid evolutionary change, but where transmission involves a biological arthropod vector, this change is constrained by the selection pressures imposed by the requirement for replication in two evolutionarily distant hosts. In this study, we use an in vivo, host-arbovirus-vector model to investigate the impact of reassortment on two phenotypic traits, virus infection rate in the vector and virulence in the host. Bluetongue virus (BTV) (Reoviridae) is the causative agent of bluetongue (BT), an economically important disease of domestic and wild ruminants and deer. The genome of BTV comprises 10 linear segments of dsRNA, and the virus is transmitted between ruminants by Culicoides biting midges (Diptera: Ceratopogonidae). Five strains of BTV representing three serotypes (BTV-1, BTV-4, and BTV-8) were isolated from naturally infected ruminants in Europe and ancestral/reassortant lineage status assigned through full genome sequencing. Each strain was then assessed in parallel for the ability to replicate in vector Culicoides and to cause BT in sheep. Our results demonstrate that two reassortment strains, which themselves became established in the field, had obtained high replication ability in C. sonorensis from one of the ancestral virus strains, which allowed inferences of the genome segments conferring this phenotypic trait. IMPORTANCE Reassortment between virus strains can lead to major shifts in the transmission parameters and virulence of segmented RNA viruses, with consequences for spread, persistence, and impact. The ability of these pathogens to adapt rapidly to their environment through this mechanism presents a major challenge in defining the conditions under which emergence can occur. Utilizing a representative mammalian host-insect vector infection and transmission model, we provide direct evidence of this phenomenon in closely related ancestral and reassortant strains of BTV. Our results demonstrate that efficient infection of Culicoides observed for one of three ancestral BTV strains was also evident in two reassortant strains that had subsequently emerged in the same ecosystem.

A Qualitative Risk Assessment for Bluetongue Disease and African Horse Sickness: The Risk of Entry and Exposure at a UK Zoo.

Bluetongue virus (BTV) and African horse sickness virus (AHSV) cause economically important diseases that are currently exotic to the United Kingdom (UK), but have significant potential for introduction and onward transmission. Given the susceptibility of animals kept in zoo collections to vector-borne diseases, a qualitative risk assessment for the introduction of BTV and AHSV to ZSL London Zoo was performed. Risk pathways for each virus were identified and assessed using published literature, animal import data and outputs from epidemiological models. Direct imports of infected animals, as well as wind-borne infected Culicoides, were considered as routes of incursion. The proximity of ongoing disease events in mainland Europe and proven capability of transmission to the UK places ZSL London Zoo at higher risk of BTV release and exposure (estimated as low to medium) than AHSV (estimated as very low to low). The recent long-range expansion of AHSV into Thailand from southern Africa highlights the need for vector competence studies of Palearctic Culicoides for AHSV to assess the risk of transmission in this region.

Experimental transmission of Leishmania (Mundinia) parasites by biting midges (Diptera: Ceratopogonidae).

Leishmania parasites, causative agents of leishmaniasis, are currently divided into four subgenera: Leishmania, Viannia, Sauroleishmania and Mundinia. The recently established subgenus Mundinia has a wide geographical distribution and contains five species, three of which have the potential to infect and cause disease in humans. While the other Leishmania subgenera are transmitted exclusively by phlebotomine sand flies (Diptera: Psychodidae), natural vectors of Mundinia remain uncertain. This study investigates the potential of sand flies and biting midges of the genus Culicoides (Diptera: Ceratopogonidae) to transmit Leishmania parasites of the subgenus Mundinia. Sand flies (Phlebotomus argentipes, P. duboscqi and Lutzomyia migonei) and Culicoides biting midges (Culicoides sonorensis) were exposed to five Mundinia species through a chicken skin membrane and dissected at specific time intervals post bloodmeal. Potentially infected insects were also allowed to feed on ear pinnae of anaesthetized BALB/c mice and the presence of Leishmania DNA was subsequently confirmed in the mice using polymerase chain reaction analyses. In C. sonorensis, all Mundinia species tested were able to establish infection at a high rate, successfully colonize the stomodeal valve and produce a higher proportion of metacyclic forms than in sand flies. Subsequently, three parasite species, L. martiniquensis, L. orientalis and L. sp. from Ghana, were transmitted to the host mouse ear by C. sonorensis bite. In contrast, transmission experiments entirely failed with P. argentipes, although colonisation of the stomodeal valve was observed for L. orientalis and L. martiniquensis and metacyclic forms of L. orientalis were recorded. This laboratory-based transmission of Mundinia species highlights that Culicoides are potential vectors of members of this ancestral subgenus of Leishmania and we suggest further studies in endemic areas to confirm their role in the lifecycles of neglected pathogens.

Thermal limits for flight activity of field-collected Culicoides in the United Kingdom defined under laboratory conditions.

BACKGROUND: Culicoides biting midges (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses and inflict biting nuisance on humans, companion animals and livestock. In temperate regions, transmission of arboviruses is limited by temperature thresholds, in both replication and dissemination of arboviruses within the vector and in the flight activity of adult Culicoides. This study aims to determine the cold-temperature thresholds for flight activity of Culicoides from the UK under laboratory conditions. METHODS: Over 18,000 Culicoides adults were collected from the field using 4 W down-draught miniature ultraviolet Centers for Disease Control traps. Populations of Culicoides were sampled at three different geographical locations within the UK during the summer months and again in the autumn at one geographical location. Activity at constant temperatures was assessed using a bioassay that detected movement of adult Culicoides towards an ultraviolet light source over a 24-h period. RESULTS: The proportion of active adult Culicoides increased with temperature but cold temperature thresholds for activity varied significantly according to collection season and location. Populations dominated by the subgenus Avaritia collected in South East England had a lower activity threshold temperature in the autumn (4 °C) compared with populations collected in the summer (10 °C). Within the subgenus Avaritia, Culicoides scoticus was significantly more active across all temperatures tested than Culicoides obsoletus within the experimental setup. Populations of Culicoides impunctatus collected in the North East of England were only active once temperatures reached 14 °C. Preliminary data suggested flight activity of the subgenus Avaritia does not differ between populations in South East England and those in the Scottish Borders. CONCLUSIONS: These findings demonstrate seasonal changes in temperature thresholds for flight and across different populations of Culicoides. These data, alongside that defining thresholds for virus replication within Culicoides, provide a primary tool for risk assessment of arbovirus transmission in temperate regions. In addition, the study also provides a comparison with thermal limits derived directly from light-suction trapping data, which is currently used as the main method to define adult Culicoides activity during surveillance.

Investigation of bovine ephemeral fever virus transmission by putative dipteran vectors under experimental conditions.

BACKGROUND: Bovine ephemeral fever virus (Rhabdoviridae: Ephemerovirus) (BEFV) causes bovine ephemeral fever (BEF), an economically important disease of cattle and water buffalo. Outbreaks of BEF in Africa, Australia, Asia and the Middle East are characterized by high rates of morbidity and highly efficient transmission between cattle hosts. Despite this, the vectors of BEFV remain poorly defined. METHODS: Colony lines of biting midges (Culicoides sonorensis) and mosquitoes (Aedes aegypti, Culex pipiens and Culex quinquefasciatus) were infected with a strain of BEFV originating from Israel by feeding on blood-virus suspensions and by intrathoracic inoculation. In addition, in vivo transmission of BEFV was also assessed by allowing C. sonorensis inoculated by the intrathoracic route to feed on male 6 month-old Holstein-Friesian calves. RESULTS: There was no evidence of BEFV replication within mosquitoes fed on blood/virus suspensions for mosquitoes of any species tested for each of the three colony lines. In 170 C. sonorensis fed on the blood/virus suspension, BEFV RNA was detected in the bodies of 13 individuals and in the heads of two individuals, indicative of fully disseminated infections and an oral susceptibility rate of 1.2%. BEFV RNA replication was further demonstrated in all C. sonorensis that were inoculated by the intrathoracic route with virus after 5, 6 or 7 days post-infection. Despite this, transmission of BEFV could not be demonstrated when infected C. sonorensis were allowed to feed on calves. CONCLUSIONS: No evidence for infection or dissemination of BEFV (bovine/Israel/2005-6) in mosquitoes of three different species was found. Evidence was found for infection of C. sonorensis by the oral route. However, attempts to transmit BEFV to calves from infected C. sonorensis failed. These results highlight the challenge of defining the natural vector of BEFV and of establishing an in vivo transmission model. The results are discussed with reference to the translation of laboratory-based studies to inference of vector competence in the field.

Correction to: The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus complex (Diptera: Ceratopogonidae) at the European level.

An amendment to this paper has been published and can be accessed via the original article.

Risk-based surveillance for bluetongue virus in cattle on the south coast of England in 2017 and 2018.

BACKGROUND: Bluetongue (BT) is a viral disease of ruminants and camelids which can have a significant impact on animal health and welfare and cause severe economic loss. The UK has been officially free of bluetongue virus (BTV) since 2011. In 2015, BTV-8 re-emerged in France and since then BTV has been spreading throughout Europe. In response to this outbreak, risk-based active surveillance was carried out at the end of the vector seasons in 2017 and 2018 to assess the risk of incursion of BTV into Great Britain. METHOD: Atmospheric dispersion modelling identified counties on the south coast of England at higher risk of an incursion. Blood samples were collected from cattle in five counties based on a sample size designed to detect at least one positive if the prevalence was 5 per cent or greater, with 95 per cent confidence. RESULTS: No virus was detected in the 478 samples collected from 32 farms at the end of the 2017 vector season or in the 646 samples collected from 43 farms at the end of the 2018 vector season, when tested by RT-qPCR. CONCLUSION: The negative results from this risk-based survey provided evidence to support the continuation of the UK's official BTV-free status.

Diversity of Transmission Outcomes Following Co-Infection of Sheep with Strains of Bluetongue Virus Serotype 1 and 8.

Bluetongue virus (BTV) causes an economically important disease, bluetongue (BT), in susceptible ruminants and is transmitted primarily by species of Culicoides biting midges (Diptera: Ceratopogonidae). Since 2006, northern Europe has experienced multiple incursions of BTV through a variety of routes of entry, including major outbreaks of strains of BTV serotype 8 (BTV-8) and BTV serotype 1 (BTV-1), which overlapped in distribution within southern Europe. In this paper, we examined the variation in response to coinfection with strains of BTV-1 and BTV-8 using an in vivo transmission model involving Culicoides sonorensis, low passage virus strains, and sheep sourced in the United Kingdom. In the study, four sheep were simultaneously infected using BTV-8 and BTV-1 intrathoracically inoculated C. sonorensis and co-infections of all sheep with both strains were established. However, there were significant variations in both the initiation and peak levels of virus RNA detected throughout the experiment, as well as in the infection rates in the C. sonorensis that were blood-fed on experimentally infected sheep at peak viremia. This is discussed in relation to the potential for reassortment between these strains in the field and the policy implications for detection of BTV strains.

Continuous Cell Lines from the European Biting Midge Culicoides nubeculosus (Meigen, 1830).

Culicoides biting midges (Diptera: Ceratopogonidae) transmit arboviruses of veterinary or medical importance, including bluetongue virus (BTV) and Schmallenberg virus, as well as causing severe irritation to livestock and humans. Arthropod cell lines are essential laboratory research tools for the isolation and propagation of vector-borne pathogens and the investigation of host-vector-pathogen interactions. Here we report the establishment of two continuous cell lines, CNE/LULS44 and CNE/LULS47, from embryos of Culicoides nubeculosus, a midge distributed throughout the Western Palearctic region. Species origin of the cultured cells was confirmed by polymerase chain reaction (PCR) amplification and sequencing of a fragment of the cytochrome oxidase 1 gene, and the absence of bacterial contamination was confirmed by bacterial 16S rRNA PCR. Both lines have been successfully cryopreserved and resuscitated. The majority of cells examined in both lines had the expected diploid chromosome number of 2n = 6. Transmission electron microscopy of CNE/LULS44 cells revealed the presence of large mitochondria within cells of a diverse population, while arrays of virus-like particles were not seen. CNE/LULS44 cells supported replication of a strain of BTV serotype 1, but not of a strain of serotype 26 which is not known to be insect-transmitted. These new cell lines will expand the scope of research on Culicoides-borne pathogens.

Outbreak of African horse sickness in Thailand, 2020.

African horse sickness was confirmed in horses in Thailand during March 2020. The virus was determined to belong to serotype 1 and is phylogenetically closely related to isolates from South Africa. This is the first incidence of African horse sickness occurring in South East Asia and of serotype 1 outside of Africa.

The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus Complex (Diptera: Ceratopogonidae) at the European level.

BACKGROUND: Culicoides obsoletus is an abundant and widely distributed Holarctic biting midge species, involved in the transmission of bluetongue virus (BTV) and Schmallenberg virus (SBV) to wild and domestic ruminants. Females of this vector species are often reported jointly with two morphologically very close species, C. scoticus and C. montanus, forming the Obsoletus/Scoticus Complex. Recently, cryptic diversity within C. obsoletus was reported in geographically distant sites. Clear delineation of species and characterization of genetic variability is mandatory to revise their taxonomic status and assess the vector role of each taxonomic entity. Our objectives were to characterize and map the cryptic diversity within the Obsoletus/Scoticus Complex. METHODS: Portion of the cox1 mitochondrial gene of 3763 individuals belonging to the Obsoletus/Scoticus Complex was sequenced. Populations from 20 countries along a Palaearctic Mediterranean transect covering Scandinavia to Canary islands (North to South) and Canary islands to Turkey (West to East) were included. Genetic diversity based on cox1 barcoding was supported by 16S rDNA mitochondrial gene sequences and a gene coding for ribosomal 28S rDNA. Species delimitation using a multi-marker methodology was used to revise the current taxonomic scheme of the Obsoletus/Scoticus Complex. RESULTS: Our analysis showed the existence of three phylogenetic clades (C. obsoletus clade O2, C. obsoletus clade dark and one not yet named and identified) within C. obsoletus. These analyses also revealed two intra-specific clades within C. scoticus and raised questions about the taxonomic status of C. montanus. CONCLUSIONS: To our knowledge, our study provides the first genetic characterization of the Obsoletus/Scoticus Complex on a large geographical scale and allows a revision of the current taxonomic classification for an important group of vector species of livestock viruses in the Palaearctic region.

Culicoides species composition and molecular identification of host blood meals at two zoos in the UK.

BACKGROUND: Culicoides biting midges are biological vectors of arboviruses including bluetongue virus (BTV), Schmallenberg virus (SBV) and African horse sickness virus (AHSV). Zoos are home to a wide range of 'at risk' exotic and native species of animals. These animals have a high value both in monetary terms, conservation significance and breeding potential. To understand the risk these viruses pose to zoo animals, it is necessary to characterise the Culicoides fauna at zoos and determine which potential vector species are feeding on which hosts. METHODS: Light-suction traps were used at two UK zoos: the Zoological Society of London (ZSL) London Zoo (LZ) and ZSL Whipsnade Zoo (WZ). Traps were run one night each week from June 2014 to June 2015. Culicoides were morphologically identified to the species level and any blood-fed Culicoides were processed for blood-meal analysis. DNA from blood meals was extracted and amplified using previously published primers. Sequencing was then carried out to determine the host species. RESULTS: A total of 11,648 Culicoides were trapped and identified (n = 5880 from ZSL WZ; n = 5768 from ZSL LZ), constituting 25 different species. The six putative vectors of BTV, SBV and AHSV in northern Europe were found at both zoos and made up the majority of the total catch (n = 10,701). A total of 31 host sequences were obtained from blood-fed Culicoides. Culicoides obsoletus/C. scoticus, Culicoides dewulfi, Culicoides parroti and Culicoides punctatus were found to be biting a wide range of mammals including Bactrian camels, Indian rhinoceros, Asian elephants and humans, with Culicoides obsoletus/C. scoticus also biting Darwin's rhea. The bird-biting species, Culicoides achrayi, was found to be feeding on blackbirds, blue tits, magpies and carrion crows. CONCLUSIONS: To our knowledge, this is the first study to directly confirm blood-feeding of Culicoides on exotic zoo animals in the UK and shows that they are able to utilise a wide range of exotic as well as native host species. Due to the susceptibility of some zoo animals to Culicoides-borne arboviruses, this study demonstrates that in the event of an outbreak of one of these viruses in the UK, preventative and mitigating measures would need to be taken.

Complete Genome Sequence of a Bovine Ephemeral Fever Virus Isolate from Israel.

Here, we report the first complete genome of a bovine ephemeral fever virus (BEFV) isolate from an infected bovine in Israel. The genome shares 95.3% identity with a Turkish genomic sequence but contains α3 and γ open reading frames that are truncated compared to those of existing BEFV genome sequences.

Long-term shifts in the seasonal abundance of adult Culicoides biting midges and their impact on potential arbovirus outbreaks.

Surveillance of adult Culicoides biting midge flight activity is used as an applied ecological method to guide the management of arbovirus incursions on livestock production in Europe and Australia.To date the impact of changes in the phenology of adult vector activity on arbovirus transmission has not been defined. We investigated this at two sites in the UK, identifying 150,000 Culicoides biting midges taken from 2867 collections over a nearly 40 year timescale.Whilst we recorded no change in seasonal activity at one site, shifts in first adult appearance and last adult appearance increased the seasonal activity period of Culicoides species at the other site by 40 days over the time period.Lengthening of the adult activity season was driven by an increase in abundance of Culicoides and correlated with local increases in temperature and precipitation. This diversity in responses poses significant challenges for predicting future transmission and overwintering risk. Policy implications. Our analysis not only shows a dramatic and consistent increase in the adult active period of Culicoides biting midges, but also that this varies significantly between sites. This suggests broad-scale analyses alone are insufficient to understand the potential impacts of changes in climate on arbovirus vector populations. Understanding the impact of climate change on adult Culicoides seasonality and transmission of arboviruses requires the context of changes in a range of other local ecological drivers.

Evidence of reduced viremia, pathogenicity and vector competence in a re-emerging European strain of bluetongue virus serotype 8 in sheep.

The outbreak of bluetongue virus (BTV) serotype 8 (BTV-8) during 2006-2009 in Europe was the most costly epidemic of the virus in recorded history. In 2015, a BTV-8 strain re-emerged in France which has continued to circulate since then. To examine anecdotal reports of reduced pathogenicity and transmission efficiency, we investigated the infection kinetics of a 2007 UK BTV-8 strain alongside the re-emerging BTV-8 strain isolated from France in 2017. Two groups of eight BTV-naïve British mule sheep were inoculated with 5.75 log10 TCID50 /ml of either BTV-8 strain. BTV RNA was detected by 2 dpi in both groups with peak viraemia occurring between 5-9 dpi. A significantly greater amount of BTV RNA was detected in sheep infected with the 2007 strain (6.0-8.8 log10 genome copies/ml) than the re-emerging BTV-8 strain (2.9-7.9 log10 genome copies/ml). All infected sheep developed BTV-specific antibodies by 9 dpi. BTV was isolated from 2 dpi to 12 dpi for 2007 BTV-8-inoculated sheep and from 5 to 10 dpi for sheep inoculated with the remerging BTV-8. In Culicoides sonorensis feeding on the sheep over the period 7-12 dpi, vector competence was significantly higher for the 2007 strain than the re-emerging strain. Both the proportion of animals showing moderate (as opposed to mild or no) clinical disease (6/8 vs. 1/8) and the overall clinical scores (median 5.25 vs. 3) were significantly higher in sheep infected with the 2007 strain, compared to those infected with the re-emerging strain. However, one sheep infected with the re-emerging strain was euthanized at 16 dpi having developed severe lameness. This highlights the potential of the re-emerging BTV-8 to still cause illness in naïve ruminants with concurrent costs to the livestock industry.

The genome of the biting midge Culicoides sonorensis and gene expression analyses of vector competence for bluetongue virus.

BACKGROUND: The new genomic technologies have provided novel insights into the genetics of interactions between vectors, viruses and hosts, which are leading to advances in the control of arboviruses of medical importance. However, the development of tools and resources available for vectors of non-zoonotic arboviruses remains neglected. Biting midges of the genus Culicoides transmit some of the most important arboviruses of wildlife and livestock worldwide, with a global impact on economic productivity, health and welfare. The absence of a suitable reference genome has hindered genomic analyses to date in this important genus of vectors. In the present study, the genome of Culicoides sonorensis, a vector of bluetongue virus (BTV) in the USA, has been sequenced to provide the first reference genome for these vectors. In this study, we also report the use of the reference genome to perform initial transcriptomic analyses of vector competence for BTV. RESULTS: Our analyses reveal that the genome is 189 Mb, assembled in 7974 scaffolds. Its annotation using the transcriptomic data generated in this study and in a previous study has identified 15,612 genes. Gene expression analyses of C. sonorensis females infected with BTV performed in this study revealed 165 genes that were differentially expressed between vector competent and refractory females. Two candidate genes, glutathione S-transferase (gst) and the antiviral helicase ski2, previously recognized as involved in vector competence for BTV in C. sonorensis (gst) and repressing dsRNA virus propagation (ski2), were confirmed in this study. CONCLUSIONS: The reference genome of C. sonorensis has enabled preliminary analyses of the gene expression profiles of vector competent and refractory individuals. The genome and transcriptomes generated in this study provide suitable tools for future research on arbovirus transmission. These provide a valuable resource for these vector lineage, which diverged from other major Dipteran vector families over 200 million years ago. The genome will be a valuable source of comparative data for other important Dipteran vector families including mosquitoes (Culicidae) and sandflies (Psychodidae), and together with the transcriptomic data can yield potential targets for transgenic modification in vector control and functional studies.